Abstract
In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx5cv mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.
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Accession codes
Abbreviations
- cDNA:
-
complementary DNA
- ChIP:
-
chromatin immunoprecipitation
- DMD:
-
Duchenne muscular dystrophy
- DNM3OS:
-
dynamin-3 opposite strand
- Dox:
-
doxycycline
- EMSA:
-
electrophoretic mobility shift assay
- FACS:
-
fluorescence-activated cell sorting
- GFP:
-
green fluorescent protein
- HRP:
-
horse radish peroxidase
- IgG:
-
immunoglobulin G
- IRES:
-
internal ribosome entry site
- MB:
-
myoblasts
- miRNA:
-
microRNA
- MOI:
-
multiplicity of infection
- MRTF:
-
myocardin-related transcription factor
- MTS:
-
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
- MSC:
-
muscle satellite cell
- MT:
-
myotubes
- mylz2:
-
myosin light chain polypeptide 2
- ORF:
-
open reading frame
- PBS:
-
phosphate-buffered saline
- qPCR:
-
quantitative PCR
- snRNA:
-
small nuclear RNA
- SRF:
-
serum response factor
- TA:
-
tibialis anterior
- TBS:
-
tris-buffered saline
- Tg:
-
transgenic
- YY1:
-
Yin-Yang 1
- WB:
-
western blot
- WNT:
-
wingless-type MMTV integration site family
- UTR:
-
untranslated region
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Acknowledgements
Funding for this work was generously provided by the Bernard F. and Alva B. Gimbel Foundation (LMK) and through a NIH grant (P50 NS040828-10). MSA was funded by a traineeship awarded by the NIH through the Harvard Stem Cell Institute and supported by a Muscular Dystrophy Association (MDA) Development Grant MDA255059. PBK is supported by the Muscular Dystrophy Association (MDA186796 and MDA 114353) and the Genise Goldenson Fund. ATK is supported by an NIH grant HL091124. The F59 (developed by F.E. Stockdale), MF20 (developed by D.A. Fischman), and Pax7 (developed by A. Kawakami) antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA, USA. We thank Louise Trakimas for assistance with the electron microscopy and Christoph Eicken at LC Sciences LLC for assistance with the microarray data analysis and processing. Flow cytometry was performed in the IDDRC Stem Cell Core Facility at Boston Children’s Hospital that is supported by National Institutes of Health award NIH-P30-HD18655. We thank Emanuela Gussoni for a critical reading of the manuscriptOur thanks to Teresa Bowman for sharing zebrafish WNT signaling primer sequences and Bryan MacDonald for helpful discussions about the WNT signaling pathway. We thank Dr. Morris White and Dr. Kyle Copps for our usage of imaging equipment. We also thank all the families and patients who donated their biopsies and time for these experiments.
Author contributions
MSA designed and performed the experiments and analyzed the data. JCC, NM, JAM, MJG, SM, and GK performed the experiments and helped in the analysis of the data. IE performed the experiments and contributed new reagents. ATK analyzed the microRNA microarray data. EAE collected patient samples, family histories, and reagents. FS isolated muscle biopsies from normal and DMD patients. PBK contributed new reagents and edited the manuscript. MSA and L.M.K. analyzed all of the data and wrote the manuscript. All authors have read and approved of the manuscript before submission.
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LMK is a consultant for Pfizer Inc. for muscle disease drug therapies. The remaining authors declare no conflict of interest.
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Alexander, M., Kawahara, G., Motohashi, N. et al. MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation. Cell Death Differ 20, 1194–1208 (2013). https://doi.org/10.1038/cdd.2013.62
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DOI: https://doi.org/10.1038/cdd.2013.62
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